DESCRIPTION: (Applicant's Description) A priority research area identified by the NCI Breast Cancer Progress Review Group is to determine how epithelial stem cells may serve as targets of important etiologic agents in breast cancer. We propose that this process critically depends upon the progressive defects in the centrosomes of stem cell. Only the division competent stem cells of the gland, as distinct from terminally differentiated cells are thought to pass on genetic damage, leading to tumors. Our morphological description of mouse mammary stem cells, and our recent application of molecular cytogenetic techniques to the study of mouse chromosomes are now combined to allow an innovative approach to the study of stem cells in mammary tumorigenesis. Specifically, the current grant will explore the mechanistic bases for alterations in mammary epithelial stem cell pathways that give rise to hyperplasias and malignant tumors in the MMTV-c-myc transgenic mammary tumor model and in the highly malignant MT-TGF-alpha X MMTV-c-myc bitransgenic model. The c-myc transgene leads initially to small, apoptotic mammary tumors that do not appear to have defects in the diversity of their stem cell pathways. However, these tumors do display amplifications in their centrosomes and alterations, such as aneuploidy, of their chromosomes. As these tumors enlarge, clearly distinct, non-apoptotic foci of cells reproducibly arise. These foci are composed primarily of the two mammary epithelial stem cell morphotypes, small light cells (SLC) and undifferentiated light cells (ULLC). The foci appear to be deficient in myoepithelial and differentiated secretory cells, consistent with further centrosomal defects, blocking their abilities to undergo the normal, asymmetric and symmetric mitoses (respectively) in the later steps of the mammary differentiation program. In contrast, bitransgenic c-Myc/TGF-alpha mammary tumors are aneuploid, but non-apoptotic from the start, and remarkably, appear to bypass the requirement for this focal block in differentiation. Our study will begin with a morphometric analysis of all cell types in the non transgenic gland and in hyperplasias and tumors of our transgenic models. We then propose to use immunohistochemistry for PCNA (proliferating cellular nuclear antigen) and labeling with a thymidine analog (BrDu) to determine the sizes of the different division-competent cell populations in normal and mouse epithelium. Next we will characterize the degree of centrosome amplification and aneuploidy in proliferative cells using immunohistochemical and FISH approaches, respectively. We expect the results of this novel proposal to demonstrate how stem cells are involved in neoplastic transformation and progression, and to suggest how different types of aberrations in the centrosomes of stem cells may be involved in causing these defects.